U.S. patent application number 13/523030 was filed with the patent office on 2013-05-02 for therapeutic polymeric nanoparticles comprising epothilone and methods of making and using same.
The applicant listed for this patent is David Dewitt, Maria Figueiredo, Young-Ho Song. Invention is credited to David Dewitt, Maria Figueiredo, Young-Ho Song.
Application Number | 20130108668 13/523030 |
Document ID | / |
Family ID | 50397493 |
Filed Date | 2013-05-02 |
United States Patent
Application |
20130108668 |
Kind Code |
A1 |
Figueiredo; Maria ; et
al. |
May 2, 2013 |
Therapeutic Polymeric Nanoparticles Comprising Epothilone and
Methods of Making and Using Same
Abstract
The present disclosure generally relates to therapeutic
nanoparticles. Exemplary nanoparticles disclosed herein may include
about 0.2 to about 20 weight percent of epothilone, e.g. epothilone
B; and about 50 to about 99 weight percent biocompatible
polymer.
Inventors: |
Figueiredo; Maria;
(Somerville, MA) ; Dewitt; David; (Allston,
MA) ; Song; Young-Ho; (Natick, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Figueiredo; Maria
Dewitt; David
Song; Young-Ho |
Somerville
Allston
Natick |
MA
MA
MA |
US
US
US |
|
|
Family ID: |
50397493 |
Appl. No.: |
13/523030 |
Filed: |
June 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US10/60575 |
Dec 15, 2010 |
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13523030 |
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61306729 |
Feb 22, 2010 |
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61405778 |
Oct 22, 2010 |
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61286550 |
Dec 15, 2009 |
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Current U.S.
Class: |
424/400 ;
514/365 |
Current CPC
Class: |
A61K 31/4178 20130101;
A61K 9/5192 20130101; A61K 9/5146 20130101; A61K 9/5153 20130101;
A61K 47/34 20130101; A61P 35/00 20180101; A61K 9/146 20130101; A61K
31/427 20130101 |
Class at
Publication: |
424/400 ;
514/365 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/4178 20060101 A61K031/4178 |
Claims
1. A therapeutic nanoparticle comprising: about 0.2 to about 20
weight percent of epothilone; and about 50 to about 99.8 weight
percent biocompatible polymer, wherein the biocompatible polymer is
selected from the group consisting of a) a diblock poly(lactic)
acid-poly(ethylene)glycol copolymer, b) a diblock
poly(lactic)-co-(glycolic) acid-poly(ethylene)glycol copolymer, c)
a combination of a) and a poly(lactic) acid homopolymer or
poly(lactic)-co-(glycolic) acid; d) a combination of b) and a
poly(lactic) acid homopolymer or poly(lactic)-co-(glycolic) acid;
e) 1,2
distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene)glycol
copolymer; and f) a combination of e) and a poly(lactic) acid
homopolymer or poly(lactic)-co-(glycolic) acid.
2. The therapeutic nanoparticle for claim 1, wherein said
epothilone is epothilone B.
3. The therapeutic nanoparticle of claim 2, comprising about 0.2 to
about 10 weight percent of epothilone.
4. The therapeutic nanoparticle of claim 1, comprising about 0.2 to
about 5 weight percent of epothilone.
5. The therapeutic nanoparticle of claim 1, wherein the diameter of
the therapeutic nanoparticle is about 60 nm to about 190 nm.
6. The therapeutic nanoparticle of claim 1, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
15 to about 90 kDa and poly(ethylene)glycol having a number average
molecular weight of about 4 to about 12 kDa.
7. The therapeutic nanoparticle of claim 1, wherein said diblock
poly(lactic)-co-glycolic acid-poly(ethylene)glycol copolymer
comprises poly(lactic acid)-co-glycolic acid having a number
average molecular weight of about 15 to about 90 kDa and
poly(ethylene)glycol having a number average molecular weight of
about 4 to about 12 kDa.
8. The therapeutic nanoparticle of claim 1, wherein the 1,2
distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene)glycol
copolymer comprises poly(ethylene)glycol having a number average
molecular weight of about 2 kDa.
9. The therapeutic nanoparticle of claim 1, wherein the particle
substantially immediately releases less than about 60% of the
therapeutic agent after 2 hours when placed in a phosphate buffer
solution at 37.degree. C.
10. The therapeutic nanoparticle of claim 1, wherein the
biocompatible polymer is diblock poly(lactic)
acid-poly(ethylene)glycol copolymer.
11. The therapeutic nanoparticle of claim 1, wherein the
therapeutic nanoparticle comprises about 40 to about 50 weight
percent diblock poly(lactic)acid-poly(ethylene)glycol copolymer and
about 40 to about 49 weight percent poly (lactic) acid
homopolymer.
12. The therapeutic nanoparticle of claim 1, wherein the poly
(lactic) acid homopolymer has a weight average molecular weight of
about 15 to about 130 kDa.
13. The therapeutic nanoparticle of claim 1, wherein the poly
(lactic) acid homopolymer has an inherent viscosity of about 0.2 to
about 0.9.
14. The therapeutic nanoparticle of claim 1, wherein the
poly(lactic) acid homopolymer has an inherent viscosity of about
0.3.
15. The therapeutic nanoparticle of claim 1, wherein the
poly(lactic) acid homopolymer has an weight average molecular
weight of about 124 kDa.
16. The therapeutic nanoparticle of claim 1, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
16 kDa and poly(ethylene)glycol having a number average molecular
weight of about 5 kDa.
17. The therapeutic nanoparticle of claim 1, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
40 to about 90 kDa and poly(ethylene)glycol having a number average
molecular weight of about 4 kDa to about 12 kDa.
18. The therapeutic nanoparticle of claim 1, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
50 kDa and poly(ethylene)glycol having a number average molecular
weight of about 5 kDa.
19. The therapeutic nanoparticle of claim 1, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
80 kDa and poly(ethylene)glycol having a number average molecular
weight of about 10 kDa.
20. The therapeutic nanoparticle of claim 1, further comprising
about 0.2 to about 10 weight percent of a diblock
poly(lactic)-poly(ethylene)glycol copolymer covalently bound to a
targeting ligand.
21. A method of treating breast, prostate, or non-small cell lung
cancer, comprising administering to a patient in need thereof an
effective amount of a composition comprising the therapeutic
nanoparticle of claim 1.
22. A plurality of therapeutic nanoparticles prepared by: combining
epothilone or pharmaceutically acceptable salts thereof and a
diblock poly(lactic)acid-polyethylene glycol or a diblock
poly(lactic)acid-co-poly(glycolic)acid-polyethylene glycol polymer
and optionally a homopolymer, with an organic solvent to form a
first organic phase having about 10 to about 40% solids; combining
the first organic phase with a first aqueous solution to form a
second phase; emulsifying the second phase to form an emulsion
phase; quenching the emulsion phase to form a quenched phase;
adding a drug solubilizer to the quenched phase to form a
solubilized phase of unencapsulated therapeutic agent; and
filtering the solubilized phase to recover the nanoparticles,
thereby forming a slurry of therapeutic nanoparticles each having
about 0.2 to about 20 weight percent of epothilone.
23. The plurality of therapeutic nanoparticles of claim 22, wherein
the epothilone is epothilone B.
24. A controlled release therapeutic nanoparticle comprising: about
0.2 to about 20 weight percent of epothilone or a pharmaceutically
acceptable salt thereof; and a diblock polymer chosen from:
poly(lactic) acid-poly(ethylene)glycol copolymer or a
poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol
copolymer, wherein said epothilone is released at a controlled
release rate.
25. The controlled release therapeutic nanoparticle of claim 24,
wherein said epothilone is epothilone B.
26. The controlled release therapeutic nanoparticle of claim 25,
wherein said epothilone is released over a period of at least 1 day
or more when administered to a patient.
27. A pharmaceutical aqueous suspension comprising a plurality of
nanoparticles of claim 1, having a glass transition temperature
between about 37.degree. C. and about 50.degree. C. in said
suspension.
28. The pharmaceutical aqueous suspension of claim 27, wherein the
glass transition temperature is between about 37.degree. C. and
about 39.degree. C.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT/US10/60575 filed
Dec. 15, 2010, which claims priority to U.S. Ser. No. 61/306,729
filed Feb. 22, 2010, U.S. Ser. No. 61/405,778 filed Oct. 22, 2010,
and U.S. Ser. No. 61/286,550 filed Dec. 15, 2009, each of which is
incorporated by reference in their entirety.
BACKGROUND
[0002] Systems that deliver certain drugs to a patient (e.g.,
targeted to a particular tissue or cell type or targeted to a
specific diseased tissue but not normal tissue), or that control
release of drugs has long been recognized as beneficial. For
example, therapeutics that include an active drug and that are
capable of locating in a particular tissue or cell type, e.g., a
specific diseased tissue, may reduce the amount of the drug in
tissues of the body that do not require treatment. This is
particularly important when treating a condition such as cancer
where it is desirable that a cytotoxic dose of the drug is
delivered to cancer cells without killing the surrounding
non-cancerous tissue. Further, such therapeutics may reduce the
undesirable and sometimes life-threatening side effects common in
anticancer therapy. For example, nanoparticle therapeutics may, due
to the small size, evade recognition within the body allowing for
targeted and controlled delivery while, e.g., remaining stable for
an effective amount of time.
[0003] Therapeutics that offer such therapy and/or controlled
release and/or targeted therapy also must be able to deliver an
effective amount of drug. It can be a challenge to prepare
nanoparticle systems that have an appropriate amount of drug
associated each nanoparticle, while keeping the size of the
nanoparticles small enough to have advantageous delivery
properties. For example, while it is desirable to load a
nanoparticle with a high quantity of therapeutic agent,
nanoparticle preparations that use a drug load that is too high
will result in nanoparticles that are too large for practical
therapeutic use. Further, it may be desirable for therapeutic
nanoparticles to remain stable so as to, e.g., substantially limit
rapid or immediate release of the therapeutic agent.
[0004] Accordingly, a need exists for new nanoparticle formulations
and methods of making such nanoparticles and compositions, that can
deliver therapeutic levels of drugs to treat diseases such as
cancer, while also reducing patient side effects. For example,
epothilone, a microtubule inhibitor with significant toxicity (e.g.
causing peripheral neuropathy), currently is administered in a
formulation having cremophor. Such formulations may result in
unwanted side effects from the active agent itself, or from
excipients with known allergic side effects.
SUMMARY
[0005] In one aspect, the invention provides therapeutic
nanoparticles that include an active agent or therapeutic agent,
e.g., epothilone (for example, epothilone B) or pharmaceutically
acceptable salts thereof, and one, two, or three biocompatible
polymers. For example, disclosed herein is a therapeutic
nanoparticle comprising about 0.2 to about 20 weight percent of
epothilone B and about 50 to about 99.8 weight percent of a
biocompatible polymer, e.g., about 70 to about 99.8 weight percent
of a biocompatible polymer. For example, the biocompatible polymer
may be a diblock poly(lactic) acid-poly(ethylene)glycol copolymer
(e.g., PLA-PEG) or a diblock (poly(lactic)-co-poly (glycolic)
acid)-poly(ethylene)glycol copolymer (e.g., PLGA-PEG), or the
biocompatible polymer may include two or more different
biocompatible polymers, for example, the therapeutic nanoparticles
can also include a homopolymer such as a poly(lactic) acid
homopolymer. For example, a disclosed therapeutic nanoparticle may
include about 0.2 to about 20 weight percent of epothilone B; and
about 50 to about 99.8 weight percent, or about 70 to about 99.8
weight percent biocompatible polymer, wherein the biocompatible
polymer is selected from the group consisting of a) a diblock
poly(lactic) acid-poly(ethylene)glycol copolymer, b) a diblock
poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol
copolymer, c) a combination of a) and a poly (lactic) acid
homopolymer; d) a combination of b) and a poly (lactic) acid
homopolymer; e) 1,2
distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene)glycol
copolymer; and f) a combination of e) and a poly (lactic) acid
homopolymer or poly(lactic)-co-(glycolic) acid.
[0006] The diameter of disclosed nanoparticles may be, for example,
about 60 to about 190 nm, about 70 to about 190 nm, about 70 to
about 180 nm or about 80 to about 180 nm.
[0007] In one embodiment, disclosed particles may substantially
release less than about 60% of the therapeutic agent over 2 hours
when placed in a phosphate buffer solution at room temperature, or
at 37.degree. C.
[0008] Epothilone may include a pharmaceutically acceptable salt
thereof. For example, contemplated nanoparticles may include about
0.2 to about 20 weight percent of epothilone B. In another example,
contemplated nanoparticles may include about 0.2 to about 15 weight
percent of epothilone B. Disclosed therapeutic nanoparticles may
include about 0.2 to about 10 weight percent epothilone B.
[0009] For example, disclosed nanoparticles may include a
biocompatible polymer that is a diblock poly(lactic)
acid-poly(ethylene)glycol copolymer. Diblock poly(lactic)
acid-poly(ethylene)glycol copolymers that may form part of a
disclosed nanoparticle may comprise poly(lactic acid) having a
number average molecular weight of about 15 to 20 kDa (or about 40
to about 90 kDa) and poly(ethylene)glycol having a number average
molecular weight of about 4 to about 6 kDa. Diblock poly(lactic)
acid-poly(ethylene)glycol copolymers that may form part of a
disclosed nanoparticle may comprise poly(lactic acid) having a
number average molecular weight of about 50 kDa and
poly(ethylene)glycol having a number average molecular weight of
about 4 to about 6 kDa. Diblock poly(lactic)-co-glycolic
acid-poly(ethylene)glycol copolymer may include poly(lactic
acid)-co-glycolic acid having a number average molecular weight of
about 15 to 20 kDa and poly(ethylene)glycol having a number average
molecular weight of about 4 to about 6 kDa. The
poly(lactic)-co-poly (glycolic) acid portion of a contemplated
diblock poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol
copolymer may have, in certain embodiments, about 50 mole percent
glycolic acid and about 50 mole percent poly(lactic) acid.
[0010] An exemplary therapeutic nanoparticle may include about 40
to about 50 weight percent diblock
poly(lactic)acid-poly(ethylene)glycol copolymer and about 40 to
about 49, or about 40 to about 60 weight percent poly (lactic) acid
homopolymer. Such poly (lactic) acid homopolymers may have e.g., a
weight average molecular weight of about 15 to about 130 kDa, e.g.,
about 10 kDa.
[0011] In an optional embodiment, a disclosed nanoparticle may
further include about 0.2 to about 10 weight percent of a diblock
poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol copolymer
covalently bound to a targeting ligand.
[0012] Also disclosed herein is a pharmaceutically acceptable
composition comprising a plurality of disclosed therapeutic
nanoparticles and a pharmaceutically acceptable excipient.
Exemplary pharmaceutically acceptable excipients may include a
sugar, such as sucrose.
[0013] For example, provided herein is a pharmaceutical aqueous
suspension comprising a plurality of nanoparticles such as those
disclosed herein, having a glass transition temperature between
about 37.degree. C. and about 50.degree. C., e.g. between about
37.degree. C. and about 39.degree. C., in the suspension.
[0014] Also disclosed herein are methods of treating cancer, such
as breast, prostate, or non-small cell lung cancer, comprising
administering to a patient in need thereof an effective amount of a
composition comprising a disclosed therapeutic nanoparticle
[0015] In another embodiment, provided herein is plurality of
therapeutic nanoparticles prepared by combining epothilone, for
example, epothilone B, or pharmaceutically acceptable salts thereof
and a diblock poly(lactic)acid-polyethylene glycol or a diblock
poly(lactic)acid-co-poly(glycolic)acid-polyethylene glycol polymer
and optionally a homopolymer, with an organic solvent to form a
first organic phase having about 10 to about 40% solids; combining
the first organic phase with a first aqueous solution to form a
second phase; emulsifying the second phase to form an emulsion
phase; quenching the emulsion phase to form a quenched phase;
adding a drug solubilizer to the quenched phase to form a
solubilized phase of unencapsulated therapeutic agent; and
filtering the solubilized phase to recover the nanoparticles,
thereby forming a slurry of therapeutic nanoparticles each having
about 0.2 to about 20 weight percent of epothilone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a flow chart for an emulsion process for forming
disclosed nanoparticles.
[0017] FIG. 2 is a flow diagram for a disclosed emulsion
process.
[0018] FIG. 3 depicts in-vitro release of epothilone B of various
nanoparticles disclosed herein.
[0019] FIG. 4 depicts the pharmokinetic profile of epothilone B
nanoparticles when administered to rats.
DETAILED DESCRIPTION
[0020] The present invention generally relates to polymeric
nanoparticles that include an active or therapeutic agent or drug,
and methods of making and using such therapeutic nanoparticles. In
general, a "nanoparticle" refers to any particle having a diameter
of less than 1000 nm, e.g. about 10 nm to about 200 nm. Disclosed
therapeutic nanoparticles may include nanoparticles having a
diameter of about 60 to about 190 nm, or about 70 to about 190 nm,
or about 60 to about 180 nm, about 70 nm to about 180 nm, or about
50 nm to about 200 nm.
[0021] Disclosed nanoparticles may include about 0.2 to about 35
weight percent, about 0.2 to about 30 weight percent, about 0.2 to
about 20 weight percent, or about 1 to about 30 weight percent of
an active agent, such as epothilone, for example, epothilone B.
[0022] Nanoparticles disclosed herein include one, two, three or
more biocompatible and/or biodegradable polymers. For example, a
contemplated nanoparticle may include about 60 to about 99.8 weight
percent of one, two, three or more biocompatible polymers such as
one or more co-polymers (e.g., a diblock polymer) that includes a
biodegradable polymer (for example, poly(lactic)acid and
polyethylene glycol) and optionally about 0 to about 50 weight
percent of a homopolymer, e.g., biodegradable polymer such as
poly(lactic) acid.
Polymers
[0023] In some embodiments, disclosed nanoparticles include a
matrix of polymers. Disclosed nanoparticles may include one or more
polymers, e.g., a diblock co-polymer and/or a monopolymer.
Disclosed therapeutic nanoparticles may include a therapeutic agent
that can be associated with the surface of, encapsulated within,
surrounded by, and/or dispersed throughout a polymeric matrix.
[0024] A wide variety of polymers and methods for forming particles
therefrom are known in the art of drug delivery. In some
embodiments, the disclosure is directed toward nanoparticles with
at least one polymer, for example, a first polymer that may be a
co-polymer, e.g., a diblock co-polymer, and optionally a polymer
that may be, for example, a homopolymer.
[0025] Any polymer can be used in accordance with the present
invention. Polymers can be natural or unnatural (synthetic)
polymers. Polymers can be homopolymers or copolymers comprising two
or more monomers. In terms of sequence, copolymers can be random,
block, or comprise a combination of random and block sequences.
Contemplated polymers may be biocompatible and/or
biodegradable.
[0026] The term "polymer," as used herein, is given its ordinary
meaning as used in the art, i.e., a molecular structure comprising
one or more repeat units (monomers), connected by covalent bonds.
The repeat units may all be identical, or in some cases, there may
be more than one type of repeat unit present within the polymer. In
some cases, the polymer can be biologically derived, i.e., a
biopolymer. Non-limiting examples include peptides or proteins. In
some cases, additional moieties may also be present in the polymer,
for example, biological moieties such as those described below. If
more than one type of repeat unit is present within the polymer,
then the polymer is said to be a "copolymer." It is to be
understood that in any embodiment employing a polymer, the polymer
being employed may be a copolymer in some cases. The repeat units
forming the copolymer may be arranged in any fashion. For example,
the repeat units may be arranged in a random order, in an
alternating order, or as a block copolymer, i.e., comprising one or
more regions each comprising a first repeat unit (e.g., a first
block), and one or more regions each comprising a second repeat
unit (e.g., a second block), etc. Block copolymers may have two (a
diblock copolymer), three (a triblock copolymer), or more numbers
of distinct blocks.
[0027] Disclosed particles can include copolymers, which, in some
embodiments, describes two or more polymers (such as those
described herein) that have been associated with each other,
usually by covalent bonding of the two or more polymers together.
Thus, a copolymer may comprise a first polymer and a second
polymer, which have been conjugated together to form a block
copolymer where the first polymer can be a first block of the block
copolymer and the second polymer can be a second block of the block
copolymer. Of course, those of ordinary skill in the art will
understand that a block copolymer may, in some cases, contain
multiple blocks of polymer, and that a "block copolymer," as used
herein, is not limited to only block copolymers having only a
single first block and a single second block. For instance, a block
copolymer may comprise a first block comprising a first polymer, a
second block comprising a second polymer, and a third block
comprising a third polymer or the first polymer, etc. In some
cases, block copolymers can contain any number of first blocks of a
first polymer and second blocks of a second polymer (and in certain
cases, third blocks, fourth blocks, etc.). In addition, it should
be noted that block copolymers can also be formed, in some
instances, from other block copolymers. For example, a first block
copolymer may be conjugated to another polymer (which may be a
homopolymer, a biopolymer, another block copolymer, etc.), to form
a new block copolymer containing multiple types of blocks, and/or
to other moieties (e.g., to non-polymeric moieties).
[0028] In some embodiments, the polymer (e.g., copolymer, e.g.,
block copolymer) can be amphiphilic, i.e., having a hydrophilic
portion and a hydrophobic portion, or a relatively hydrophilic
portion and a relatively hydrophobic portion. A hydrophilic polymer
can be one that generally that attracts water and a hydrophobic
polymer can be one that generally repels water. A hydrophilic or a
hydrophobic polymer can be identified, for example, by preparing a
sample of the polymer and measuring its contact angle with water
(typically, the polymer will have a contact angle of less than
60.degree., while a hydrophobic polymer will have a contact angle
of greater than about 60.degree.). In some cases, the
hydrophilicity of two or more polymers may be measured relative to
each other, i.e., a first polymer may be more hydrophilic than a
second polymer. For instance, the first polymer may have a smaller
contact angle than the second polymer.
[0029] In one set of embodiments, a polymer (e.g., copolymer, e.g.,
block copolymer) contemplated herein includes a biocompatible
polymer, i.e., the polymer that does not typically induce an
adverse response when inserted or injected into a living subject,
for example, without significant inflammation and/or acute
rejection of the polymer by the immune system, for instance, via a
T-cell response. Accordingly, the therapeutic particles
contemplated herein can be non-immunogenic. The term
"non-immunogenic" as used herein refers to endogenous growth factor
in its native state which normally elicits no, or only minimal
levels of, circulating antibodies, T-cells, or reactive immune
cells, and which normally does not elicit in the individual an
immune response against itself.
[0030] Biocompatibility typically refers to the acute rejection of
material by at least a portion of the immune system, i.e., a
nonbiocompatible material implanted into a subject provokes an
immune response in the subject that can be severe enough such that
the rejection of the material by the immune system cannot be
adequately controlled, and often is of a degree such that the
material must be removed from the subject. One simple test to
determine biocompatibility can be to expose a polymer to cells in
vitro; biocompatible polymers are polymers that typically will not
result in significant cell death at moderate concentrations, e.g.,
at concentrations of 50 micrograms/10.sup.6 cells. For instance, a
biocompatible polymer may cause less than about 20% cell death when
exposed to cells such as fibroblasts or epithelial cells, even if
phagocytosed or otherwise uptaken by such cells. Non-limiting
examples of biocompatible polymers that may be useful in various
embodiments of the present invention include polydioxanone (PDO),
polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate),
polyglycolide, polylactide, PLGA, polycaprolactone, or copolymers
or derivatives including these and/or other polymers.
[0031] In certain embodiments, contemplated biocompatible polymers
may be biodegradable, i.e., the polymer is able to degrade,
chemically and/or biologically, within a physiological environment,
such as within the body. As used herein, "biodegradable" polymers
are those that, when introduced into cells, are broken down by the
cellular machinery (biologically degradable) and/or by a chemical
process, such as hydrolysis, (chemically degradable) into
components that the cells can either reuse or dispose of without
significant toxic effect on the cells. In one embodiment, the
biodegradable polymer and their degradation byproducts can be
biocompatible.
[0032] For instance, a contemplated polymer may be one that
hydrolyzes spontaneously upon exposure to water (e.g., within a
subject), or the polymer may degrade upon exposure to heat (e.g.,
at temperatures of about 37.degree. C.). Degradation of a polymer
may occur at varying rates, depending on the polymer or copolymer
used. For example, the half-life of the polymer (the time at which
50% of the polymer can be degraded into monomers and/or other
nonpolymeric moieties) may be on the order of days, weeks, months,
or years, depending on the polymer. The polymers may be
biologically degraded, e.g., by enzymatic activity or cellular
machinery, in some cases, for example, through exposure to a
lysozyme (e.g., having relatively low pH).
[0033] In some cases, the polymers may be broken down into monomers
and/or other nonpolymeric moieties that cells can either reuse or
dispose of without significant toxic effect on the cells (for
example, polylactide may be hydrolyzed to form lactic acid,
polyglycolide may be hydrolyzed to form glycolic acid, etc.).
[0034] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEGylated polymers and copolymers of lactide and
glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA, and
derivatives thereof). In some embodiments, polyesters include, for
example, polyanhydrides, poly(ortho ester), PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated
poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine
ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0035] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA can be characterized by
the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid-glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention can be characterized by a lactic acid:glycolic
acid molar ratio of approximately 85:15, approximately 75:25,
approximately 60:40, approximately 50:50, approximately 40:60,
approximately 25:75, or approximately 15:85.
[0036] In some embodiments, the ratio of lactic acid to glycolic
acid monomers in the polymer of the particle (e.g., the PLGA block
copolymer or PLGA-PEG block copolymer) may be selected to optimize
for various parameters such as water uptake, therapeutic agent
release and/or polymer degradation kinetics.
[0037] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, amino alkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid)
polyacrylamide, amino alkyl methacrylate copolymer, glycidyl
methacrylate copolymers, polycyanoacrylates, and combinations
comprising one or more of the foregoing polymers. The acrylic
polymer may comprise fully-polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0038] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g., DNA, RNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine), polyethylene imine (PEI), and poly(amidoamine)
dendrimers are contemplated for use, in some embodiments, in a
disclosed particle.
[0039] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains. Examples of these polyesters include
poly(L-lactide-co-L-lysine), poly(serine ester), and
poly(4-hydroxy-L-proline ester). A polymer (e.g., copolymer, e.g.,
block copolymer) containing poly(ethylene glycol) repeat units can
also be referred to as a "PEGylated" polymer. Such polymers can
control inflammation and/or immunogenicity (i.e., the ability to
provoke an immune response) and/or lower the rate of clearance from
the circulatory system via the reticuloendothelial system (RES) due
to the presence of the poly(ethylene glycol) groups.
[0040] PEGylation may also be used, in some cases, to decrease
charge interaction between a polymer and a biological moiety, e.g.,
by creating a hydrophilic layer on the surface of the polymer,
which may shield the polymer from interacting with the biological
moiety. In some cases, the addition of poly(ethylene glycol) repeat
units may increase plasma half-life of the polymer (e.g.,
copolymer, e.g., block copolymer), for instance, by decreasing the
uptake of the polymer by the phagocytic system while decreasing
transfection/uptake efficiency by cells. Those of ordinary skill in
the art will know of methods and techniques for PEGylating a
polymer, for example, by using EDC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and
NHS (N-hydroxysuccinimide) to react a polymer to a PEG group
terminating in an amine, by ring opening polymerization techniques
(ROMP), or the like.
[0041] It is contemplated that PEG may include a terminal end
group, for example, when PEG is not conjugated to a ligand. For
example, PEG may terminate in a hydroxyl, a methoxy or other
alkoxyl group, a methyl or other alkyl group, an aryl group, a
carboxylic acid, an amine, an amide, an acetyl group, a guanidino
group, or an imidazole. Other contemplated end groups include
azide, alkyne, maleimide, aldehyde, hydrazide, hydroxylamine,
alkoxyamine, or thiol moieties.
[0042] Particles disclosed herein may or may not contain PEG. In
addition, certain embodiments can be directed towards copolymers
containing poly(ester-ether).sub.s, e.g., polymers having repeat
units joined by ester bonds (e.g., R--C(O)--O--R' bonds) and ether
bonds (e.g., R--O--R' bonds). In some embodiments of the invention,
a biodegradable polymer, such as a hydrolyzable polymer, containing
carboxylic acid groups may be conjugated with poly(ethylene glycol)
repeat units to form a poly(ester-ether).
[0043] In one embodiment, the molecular weight of the polymers can
be optimized for effective treatment as disclosed herein. For
example, the molecular weight of a polymer may influence particle
degradation rate (such as when the molecular weight of a
biodegradable polymer can be adjusted), solubility, water uptake,
and drug release kinetics. For example, the molecular weight of the
polymer can be adjusted such that the particle biodegrades in the
subject being treated within a reasonable period of time (ranging
from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks,
etc.). A disclosed particle can for example comprise a copolymer of
PEG and PLA, the PEG can have a molecular weight of 1,000-20,000
Da, e.g., 5,000-20,000 Da, e.g., 10,000-20,000 Da, and the PLA or
PEG-PLA can have a molecular weight of 5,000-100,000 Da, e.g.,
20,000-70,000 Da, e.g., 15,000-50,000 Da.
[0044] For example, disclosed herein is an exemplary therapeutic
nanoparticle that includes about 10 to about 99 weight percent
poly(lactic) acid-poly(ethylene)glycol copolymer or
poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol
copolymer, or about 20 to about 80 weight percent, about 40 to
about 80 weight percent, or about 30 to about 50 weight percent, or
about 70 to about 90 weight percent poly(lactic)
acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly
(glycolic) acid-poly(ethylene)glycol copolymer. Exemplary
poly(lactic) acid-poly(ethylene)glycol copolymers can include a
number average molecular weight of about or about 10 to about 90
kDa, or about 15 to about 20 kDa, or about 10 to about 25 kDa of
poly(lactic) acid, or about 40 kDa to about 90 kDa, or about 50 kDa
to about 80 kDa, and a number average molecular weight of about 4
to about 6 kDa, about 4 to about 12 kDa, or about 2 to about 10 kDa
of poly(ethylene)glycol.
[0045] Disclosed nanoparticles may optionally include about 1 to
about 50 weight percent poly(lactic) acid or poly(lactic)
acid-co-poly (glycolic) acid (which does not include PEG, e.g., a
homopolymer of PLA), or may optionally include about 1 to about 50
(or about 1 to about 70) weight percent, or about 10 to about 50
weight percent, or about 30 to about 50 weight percent poly(lactic)
acid or poly(lactic) acid-co-poly (glycolic) acid. In an
embodiment, disclosed nanoparticles may include two polymers, e.g.
PLA-PEG and PLA, in a weight ratio of about 40:60 to about 60:40,
about 50:30 to about 30:50, e.g., about 50:50 (PLA-PEG to PLA).
[0046] Such substantially homopolymeric poly(lactic) or
poly(lactic)-co-poly(glycolic) acid may have a weight average
molecular weight of about 4.5 to about 130 kDa, for example, about
20 to about 30 kDa, or about 100 to about 130 kDa. Such
homopolymeric PLA may have a number average molecule weight of
about 4.5 to about 90 kDa, or about 4.5 to about 12 kDa, about 5.5
to about 7 kDa (e.g. about 6.5 kDa), about 15 to about 30 kDa, or
about 60 to about 90 kDa. Exemplary homopolymeric PLA may have a
number average molecular weight of about 70 or 80 kDa or a weight
average molecular weight of about 124 kD. As is known in the art,
molecular weight of polymers can be related to an inherent
viscosity. In some embodiments, homopolymer PLA may have an
inherent viscosity of about 0.2 to about 0.4, e.g. about 0.4; in
other embodiments, PLA may have an inherent viscosity of about 0.6
to about 0.8. Exemplary PLGA may have a number average molecular
weight of about 8 to about 12 kDa.
[0047] In certain embodiments, disclosed polymers may be conjugated
to a lipid, e.g., "end-capped," for example, may include a
lipid-terminated PEG. As described below, the lipid portion of the
polymer can be used for self-assembly with another polymer,
facilitating the formation of a nanoparticle. For example, a
hydrophilic polymer could be conjugated to a lipid that will self
assemble with a hydrophobic polymer.
[0048] Exemplary lipids include fatty acids such as long chain
(e.g., C.sub.8-C.sub.50), substituted or unsubstituted
hydrocarbons. In some embodiments, a fatty acid group can be a
C.sub.10-C.sub.20 fatty acid or salt thereof. In some embodiments,
a fatty acid group can be a C.sub.15-C.sub.20 fatty acid or salt
thereof. In some embodiments, a fatty acid can be unsaturated,
monounsaturated, or polyunsaturated. For example, a fatty acid
group can be one or more of butyric, caproic, caprylic, capric,
lauric, myristic, palmitic, stearic, arachidic, behenic, or
lignoceric acid. In some embodiments, a fatty acid group can be one
or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0049] In a particular embodiment, the lipid is of the Formula
V:
##STR00001##
and salts thereof, wherein each R is, independently, C.sub.1-30
alkyl. In one embodiment of Formula V, the lipid is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof, e.g., the sodium salt. For example, DSPE may be conjugated
to PEG via the --NH moiety.
[0050] In one embodiment, optional small molecule targeting
moieties are bonded, e.g., covalently bonded, to the lipid
component of the nanoparticle. For example, contemplated herein is
also a nanoparticle comprising a therapeutic agent, a polymeric
matrix comprising functionalized and non-functionalized polymers, a
lipid, and a low-molecular weight targeting ligand, wherein the
targeting ligand is bonded, e.g., covalently bonded, to the lipid
component of the nanoparticle.
Targeting Moieties
[0051] Provided herein are nanoparticles that may include an
optional targeting moiety, i.e., a moiety able to bind to or
otherwise associate with a biological entity, for example, a
membrane component, a cell surface receptor, prostate specific
membrane antigen, or the like. A targeting moiety present on the
surface of the particle may allow the particle to become localized
at a particular targeting site, for instance, a tumor, a disease
site, a tissue, an organ, a type of cell, etc. The drug or other
payload may then, in some cases, be released from the particle and
allowed to interact locally with the particular targeting site.
[0052] In one embodiment of the instant invention, the targeting
moiety may be a low-molecular weight ligand, e.g., a low-molecular
weight PSMA ligand. For example, a targeting portion may cause the
particles to become localized to a tumor, a disease site, a tissue,
an organ, a type of cell, etc. within the body of a subject,
depending on the targeting moiety used. For example, a
low-molecular weight PSMA ligand may become localized to prostate
cancer cells. The subject may be a human or non-human animal.
Examples of subjects include, but are not limited to, a mammal such
as a dog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a
sheep, a goat, a rat, a mouse, a guinea pig, a hamster, a primate,
a human or the like.
[0053] Contemplated targeting moieties include small molecules. In
certain embodiments, the term "small molecule" refers to organic
compounds, whether naturally-occurring or artificially created
(e.g., via chemical synthesis) that have relatively low molecular
weight and that are not proteins, polypeptides, or nucleic acids.
Small molecules typically have multiple carbon-carbon bonds. In
certain embodiments, small molecules are less than about 2000 g/mol
in size. In some embodiments, small molecules are less than about
1500 g/mol or less than about 1000 g/mol. In some embodiments,
small molecules are less than about 800 g/mol or less than about
500 g/mol, for example about 100 g/mol to about 600 g/mol, or about
200 g/mol to about 500 g/mol. For example, a ligand may be a
low-molecular weight PSMA ligand such as
##STR00002##
and enantiomers, stereoisomers, rotamers, tautomers, diastereomers,
or racemates thereof.
[0054] In some embodiments, small molecule targeting moieties that
may be used to target cells associated with prostate cancer tumors
include PSMA peptidase inhibitors such as 2-PMPA, GPI5232, VA-033,
phenylalkylphosphonamidates and/or analogs and derivatives thereof.
In some embodiments, small molecule targeting moieties that may be
used to target cells associated with prostate cancer tumors include
thiol and indole thiol derivatives, such as 2-MPPA and
3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives. In
some embodiments, small molecule targeting moieties that may be
used to target cells associated with prostate cancer tumors include
hydroxamate derivatives. In some embodiments, small molecule
targeting moieties that may be used to target cells associated with
prostate cancer tumors include PBDA- and urea-based inhibitors,
such as ZJ 43, ZJ 11, ZJ 17, ZJ 38 and/or analogs and derivatives
thereof, androgen receptor targeting agents (ARTAs), polyamines,
such as putrescine, spermine, and spermidine, and inhibitors of the
enzyme glutamate carboxylase II (GCPII), also known as NAAG
Peptidase or NAALADase.
[0055] Contemplated targeting moieties include peptides. Peptides
are typically below 40 amino acids in length. Examples of peptide
lengths include peptides of 2 amino acids, 3 amino acids, 4 amino
acids, 5 amino acids, 5-10 amino acids, 7-15 amino acids, 10-20
amino acids, 15-25 amino acids, 15-30 amino acids, 5-40 amino
acids, and 25-40 amino acids.
[0056] In another embodiment of the instant invention, the
targeting moiety can be a ligand that targets Her2, EGFR, or toll
receptors. For example, contemplated targeting moieties may include
a nucleic acid, polypeptide, glycoprotein, carbohydrate, or lipid.
For example, a targeting moiety can be a nucleic acid targeting
moiety (e.g., an aptamer, e.g., the A10 aptamer) that binds to a
cell type specific marker. In general, an aptamer is an
oligonucleotide (e.g., DNA, RNA, or an analog or derivative
thereof) that binds to a particular target, such as a polypeptide.
In some embodiments, a targeting moiety may be a naturally
occurring or synthetic ligand for a cell surface receptor, e.g., a
growth factor, hormone, LDL, transferrin, etc. A targeting moiety
can be an antibody, which term is intended to include antibody
fragments. Characteristic portions of antibodies, such as single
chain targeting moieties, can be identified, e.g., using procedures
such as phage display. Targeting moieties may be a targeting
peptide or targeting peptidomimetic that has a length of up to
about 50 residues. For example, targeting moieties may include the
amino acid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X
and Z are variable amino acids, or conservative variants or
peptidomimetics thereof. In particular embodiments, the targeting
moiety is a peptide that includes the amino acid sequence AKERC,
CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino
acids, and has a length of less than 20, 50 or 100 residues. The
CREKA (Cys Arg Glu Lys Ala) peptide or a peptidomimetic thereof or
the octapeptide AXYLZZLN are also contemplated as targeting
moieties, as well as peptides, or conservative variants or
peptidomimetics thereof, that bind or form a complex with collagen
IV, or that target tissue basement membrane (e.g., the basement
membrane of a blood vessel).
[0057] Exemplary targeting moieties include peptides that target
ICAM (intercellular adhesion molecule, e.g., ICAM-1).
[0058] Targeting moieties disclosed herein are typically conjugated
to a disclosed polymer or copolymer (e.g., PLA-PEG), and such a
polymer conjugate may form part of a disclosed nanoparticle. For
example, a disclosed therapeutic nanoparticle may optionally
include about 0.2 to about 10 weight percent of a PLA-PEG or
PLGA-PEG, wherein the PEG is functionalized with a targeting
ligand. Contemplated therapeutic nanoparticles may include, for
example, about 0.2 to about 10 mole percent PLA-PEG-ligand or poly
(lactic) acid-co-poly (glycolic) acid-PEG-ligand. For example,
PLA-PEG-ligand may include a PLA with a number average molecular
weight of about 10 kDa to about 20 kDa and PEG with a number
average molecular weight of about 4,000 to about 8,000 Da.
Nanoparticles
[0059] Disclosed nanoparticles may have a substantially spherical
(i.e., the particles generally appear to be spherical), or
non-spherical configuration. For instance, the particles, upon
swelling or shrinkage, may adopt a non-spherical configuration. In
some cases, the particles may include polymeric blends. For
instance, a polymer blend may include a first co-polymer that
includes polyethylene glycol and a second polymer.
[0060] Disclosed nanoparticles may have a characteristic dimension
of less than about 1 micrometer, where the characteristic dimension
of a particle is the diameter of a perfect sphere having the same
volume as the particle. For example, the particle can have a
characteristic dimension of the particle less than about 300 nm,
less than about 200 nm, less than about 150 nm, less than about 100
nm, less than about 50 nm, less than about 30 nm, less than about
10 nm, less than about 3 nm, or less than about 1 nm in some cases.
In particular embodiments, disclosed nanoparticles may have a
diameter of about 60 nm to about 200 nm, about 60 nm to about 190
nm, about 70 nm to about 180 nm, or about 80 nm to about 180
nm.
[0061] In one set of embodiments, the particles can have an
interior and a surface, where the surface has a composition
different from the interior, i.e., there may be at least one
compound present in the interior but not present on the surface (or
vice versa), and/or at least one compound is present in the
interior and on the surface at differing concentrations. For
example, in one embodiment, a compound, such as a targeting moiety
(i.e., a low-molecular weight ligand) of a polymeric conjugate of
the present invention, may be present in both the interior and the
surface of the particle, but at a higher concentration on the
surface than in the interior of the particle. Although in some
cases, the concentration in the interior of the particle may be
essentially nonzero, i.e., there is a detectable amount of the
compound present in the interior of the particle.
[0062] In some cases, the interior of the particle is more
hydrophobic than the surface of the particle. For instance, the
interior of the particle may be relatively hydrophobic with respect
to the surface of the particle, and a drug or other payload may be
hydrophobic, and readily associates with the relatively hydrophobic
center of the particle. The drug or other payload can thus be
contained within the interior of the particle, which can shelter it
from the external environment surrounding the particle (or vice
versa). For instance, a drug or other payload contained within a
particle administered to a subject will be protected from a
subject's body, and the body may also be substantially isolated
from the drug for at least a period of time.
[0063] For example, disclosed herein is a therapeutic polymeric
nanoparticle comprising a first non-functionalized polymer; an
optional second non-functionalized polymer; an optional
functionalized polymer comprising a targeting moiety; and a
therapeutic agent. In a particular embodiment, the first
non-functionalized polymer is PLA, PLGA, or PEG, or copolymers
thereof, e.g., a diblock co-polymer PLA-PEG. For example, exemplary
nanoparticles may have a PEG corona with a density of about 0.065
g/cm.sup.3, or about 0.01 to about 0.10 g/cm.sup.3.
[0064] Disclosed nanoparticles may be stable, for example in a
solution that may contain a saccharide, e.g., sugar, for at least
about 3 days, at least about 4 days or at least about 5 days at
room temperature, or at 25.degree. C.
[0065] In some embodiments, disclosed nanoparticles may also
include a fatty alcohol, which may increase the rate of drug
release. For example, disclosed nanoparticles may include a
C.sub.8-C.sub.30 alcohol such as cetyl alcohol, octanol, stearyl
alcohol, arachidyl alcohol, docosonal, or octasonal.
[0066] Nanoparticles may have controlled release properties, e.g.,
may be capable of delivering an amount of active agent to a
patient, e.g., to specific site in a patient, over an extended
period of time, e.g. over 1 day, 1 week, or more. In some
embodiments, disclosed nanoparticles substantially immediately
release (e.g., over about 1 minute to about 30 minutes) less than
about 2%, less than about 4%, less than about 5%, or less than
about 10% of an active agent (e.g. epothilone B), for example when
placed in a phosphate buffer solution at room temperature and/or at
37.degree. C.
[0067] In another embodiment, a disclosed nanoparticle may release
less than about 20%, less than about 30%, less than about 40%, less
than 50%, or even less than 60% (or more) for example when placed
in a phosphate buffer solution at room temperature or at 37.degree.
C., for 1 day or more. In one embodiment, a disclosed nanoparticle
may release less than about 60% of the therapeutic agent over 2
hours when placed in a phosphate buffer solution at room
temperature.
[0068] In one embodiment, the invention comprises a nanoparticle
comprising 1) a polymeric matrix and 2) an amphiphilic compound or
layer that surrounds or is dispersed within the polymeric matrix
forming a continuous or discontinuous shell for the particle. An
amphiphilic layer can reduce water penetration into the
nanoparticle, thereby enhancing drug encapsulation efficiency and
slowing drug release. Further, these amphiphilic layer protected
nanoparticles can provide therapeutic advantages by releasing the
encapsulated drug and polymer at appropriate times.
[0069] As used herein, the term "amphiphilic" refers to a property
where a molecule has both a polar portion and a non-polar portion.
Often, an amphiphilic compound has a polar head attached to a long
hydrophobic tail. In some embodiments, the polar portion is soluble
in water, while the non-polar portion is insoluble in water. In
addition, the polar portion may have either a formal positive
charge, or a formal negative charge. Alternatively, the polar
portion may have both a formal positive and a negative charge, and
be a zwitterion or inner salt. Exemplary amphiphilic compound
include, for example, one or a plurality of the following:
naturally derived lipids, surfactants, or synthesized compounds
with both hydrophilic and hydrophobic moieties.
[0070] Specific examples of amphiphilic compounds include, but are
not limited to, phospholipids, such as 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcholine (DBPC),
ditricosanoylphosphatidylcholine (DTPC), and
dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of
between 0.01-60 (weight lipid/weight polymer), most preferably
between 0.1-30 (weight lipid/weight polymer). Phospholipids which
may be used include, but are not limited to, phosphatidic acids,
phosphatidyl cholines with both saturated and unsaturated lipids,
phosphatidyl ethanolamines, phosphatidylglycerols,
phosphatidylserines, phosphatidylinositols, lysophosphatidyl
derivatives, cardiolipin, and .beta.-acyl-y-alkyl phospholipids.
Examples of phospholipids include, but are not limited to,
phosphatidylcholines such as dioleoylphosphatidylcholine,
dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine
dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine
(DPPC), distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcho-line (DBPC),
ditricosanoylphosphatidylcholine (DTPC),
dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines
such as dioleoylphosphatidylethanolamine or
1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Synthetic
phospholipids with asymmetric acyl chains (e.g., with one acyl
chain of 6 carbons and another acyl chain of 12 carbons) may also
be used.
[0071] In a particular embodiment, an amphiphilic component may
include lecithin, and/or in particular, phosphatidylcholine.
Preparation of Nanoparticles
[0072] Another aspect of the invention is directed to systems and
methods of making disclosed nanoparticles. In some embodiments, by
using two or more different polymers (e.g., a copolymer such as a
diblock copolymer and a homopolymer) properties of particles may be
controlled.
[0073] In a particular embodiment, the methods described herein
form nanoparticles that have a high amount of encapsulated
therapeutic agent, for example, that may include about 0.2 to about
40 weight percent, or about 0.2 to about 30 weight percent, e.g.,
about 0.2 to about 20 weight percent or about 1 to about 10 weight
percent epothilone B.
[0074] In an embodiment, a nanoemulsion process is provided, such
as the process represented in FIGS. 1 and 2. For example, a
therapeutic agent, a first polymer (for example, PLA-PEG or
PLGA-PEG) and/or a second polymer (e.g. (PL(G)A or PLA), is mixed
with an organic solution to form a first organic phase. Such first
phase may include about 5 to about 50% weight solids, e.g. about 5
to about 40% solids, or about 10 to about 30% solids, e.g. about
10%, 15%, 20% solids. The first organic phase may be combined with
a first aqueous solution to form a second phase. The organic
solution can include, for example, acetonitrile, tetrahydrofuran,
ethyl acetate, isopropyl alcohol, isopropyl acetate,
dimethylformamide, methylene chloride, dichloromethane, chloroform,
acetone, benzyl alcohol, Tween 80, Span 80, or the like, and
combinations thereof. In an embodiment, the organic phase may
include benzyl alcohol, ethyl acetate, and combinations thereof.
The second phase can be between about 1 and 50 weight %, e.g., 5-40
weight %, solids. The aqueous solution can be water, optionally in
combination with one or more of sodium cholate, ethyl acetate, and
benzyl alcohol.
[0075] For example, the oil or organic phase may use solvent that
is only partially miscible with the nonsolvent (water). Therefore,
when mixed at a low enough ratio and/or when using water
pre-saturated with the organic solvents, the oil phase remains
liquid. The oil phase may be emulsified into an aqueous solution
and, as liquid droplets, sheared into nanoparticles using, for
example, high energy dispersion systems, such as homogenizers or
sonicators. The aqueous portion of the emulsion, otherwise known as
the "water phase", may be surfactant solution consisting of sodium
cholate and pre-saturated with ethyl acetate and benzyl
alcohol.
[0076] Emulsifying the second phase to form an emulsion phase may
be performed in one or two emulsification steps. For example, a
primary emulsion may be prepared, and then emulsified to form a
fine emulsion. The primary emulsion can be formed, for example,
using simple mixing, a high pressure homogenizer, probe sonicator,
stir bar, or a rotor stator homogenizer. The primary emulsion may
be formed into a fine emulsion through the use of e.g. probe
sonicator or a high pressure homogenizer, e.g. by using 1, 2, 3 or
more passes through a homogenizer. For example, when a high
pressure homogenizer is used, the pressure used may be about 5000
to about 15000 psi, or about 9900 to about 13200 psi, e.g. 9900 or
13200 psi.
[0077] Either solvent evaporation or dilution may be needed to
complete the extraction of the solvent and solidify the particles.
For better control over the kinetics of extraction and a more
scalable process, a solvent dilution via aqueous quench may be
used. For example, the emulsion can be diluted into cold water to a
concentration sufficient to dissolve all of the organic solvent to
form a quenched phase. Quenching may be performed at least
partially at a temperature of about 5.degree. C. or less. For
example, water used in the quenching may be at a temperature that
is less that room temperature (e.g. about 0 to about 10.degree. C.,
or about 0 to about 5.degree. C.).
[0078] In some embodiments, not all of the therapeutic agent is
encapsulated in the particles at this stage, and a drug solubilizer
is added to the quenched phase to form a solubilized phase. The
drug solubilizer may be for example, Tween 80, Tween 20, polyvinyl
pyrrolidone, cyclodextran, sodium dodecyl sulfate, or sodium
cholate. For example, Tween-80 may added to the quenched
nanoparticle suspension to solubilize the free drug and prevent the
formation of drug crystals. In some embodiments, a ratio of drug
solubilizer to therapeutic agent is about 100:1 to about 10:1.
[0079] The solubilized phase may be filtered to recover the
nanoparticles. For example, ultrafiltration membranes may be used
to concentrate the nanoparticle suspension and substantially
eliminate organic solvent, free drug, and other processing aids
(surfactants). Exemplary filtration may be performed using a
tangential flow filtration system. For example, by using a membrane
with a pore size suitable to retain nanoparticles while allowing
solutes, micelles, and organic solvent to pass, nanoparticles can
be selectively separated. Exemplary membranes with molecular weight
cut-offs of about 300-500 kDa (.about.5-25 nm) may be used.
[0080] Diafiltration may be performed using a constant volume
approach, meaning the diafiltrate (cold deionized water, e.g. about
0.degree. C. to about 5.degree. C., or 0 to about 10.degree. C.)
may added to the feed suspension at the same rate as the filtrate
is removed from the suspension. In some embodiments, filtering may
include a first filtering using a first temperature of about
0.degree. C. to about 5.degree. C., or 0.degree. C. to about
10.degree. C., and optionally a second temperature of about
20.degree. C. to about 30.degree. C., or 15.degree. C. to about
35.degree. C. For example, filtering may include processing about
10 to about 20 diavolumes at about 0.degree. C. to about 5.degree.
C. In another embodiment, filtering may include processing about 1
to about 6 diavolumes at about 0.degree. C. to about 5.degree. C.,
and processing at least one diavolume (e.g. about 1 to about 3 or
about 1-2 diavolumes) at about 20.degree. C. to about 30.degree.
C.
[0081] Optionally, after purifying and concentrating the
nanoparticle suspension, the particles may be passed through one,
two or more sterilizing and/or depth filters, for example, using
.about.0.2 .mu.m depth pre-filter.
[0082] In exemplary embodiment of preparing nanoparticles, an
organic phase is formed composed of a mixture of a therapeutic
agent, e.g., epothilone B, and polymer (homopolymer, and
co-polymer). The organic phase may be mixed with an aqueous phase
at approximately a 1:5 ratio (oil phase:aqueous phase) where the
aqueous phase is composed of a surfactant and optionally dissolved
solvent. A primary emulsion may then formed by the combination of
the two phases under simple mixing or through the use of a rotor
stator homogenizer. The primary emulsion is then formed into a fine
emulsion through the use of e.g. high pressure homogenizer. Such
fine emulsion may then quenched by, e.g. addition to deionized
water under mixing. An exemplary quench:emulsion ratio may be about
approximately 8:1. A solution of Tween (e.g., Tween 80) can then be
added to the quench to achieve e.g. approximately 1-2% Tween
overall, which may serve to dissolve free, unencapsulated drug.
Formed nanoparticles may then be isolated through either
centrifugation or ultrafiltration/diafiltration.
Therapeutic Agents
[0083] In a particular embodiment, a therapeutic agent or drug,
e.g., epothilone B, may be released in a controlled release manner
from the particle and allowed to interact locally with the
particular patient site (e.g., a tumor). The term "controlled
release" is generally meant to encompass release of a substance
(e.g., a drug) at a selected site or otherwise controllable in
rate, interval, and/or amount. Controlled release encompasses, but
is not necessarily limited to, substantially continuous delivery,
patterned delivery (e.g., intermittent delivery over a period of
time that is interrupted by regular or irregular time intervals),
and delivery of a bolus of a selected substance (e.g., as a
predetermined, discrete amount if a substance over a relatively
short period of time (e.g., a few seconds or minutes)).
[0084] The active agent or drug may be an epothilone such as
epothilone A, B, C, D, E, F or a pharmaceutically acceptable salt
thereof. For example, the active agent or drug may be epothilone B.
Contemplated epothilone compounds include dehydelone, ixabepilone,
and sagopilone.
[0085] In an embodiment, an active agent may (or in another
embodiment, may not be) conjugated to e.g. a disclosed hydrophobic
polymer that forms part of a disclosed nanoparticle, e.g. an active
agent such as epothilone may be conjugated (e.g. covalently bound,
e.g. directly or through a linking moiety such as linking moiety
comprising e.g., --NH-alkylene-C(O)--,
--NH-alkylene-O-alkylene-C(O)--,
--NH-alkylene-C(O)--O-alkylene-C(O)--, or --NH-alkylene-S--) to PLA
or PGLA, or a PLA or PLGA portion of a copolymer such as PLA-PEG or
PLGA-PEG.
Pharmaceutical Formulations
[0086] Nanoparticles disclosed herein may be combined with
pharmaceutical acceptable carriers to form a pharmaceutical
composition. As would be appreciated by one of skill in this art,
the carriers may be chosen based on the route of administration as
described below, the location of the target issue, the drug being
delivered, the time course of delivery of the drug, etc.
[0087] The pharmaceutical compositions and particles disclosed
herein can be administered to a patient by any means known in the
art including oral and parenteral routes. The term "patient," as
used herein, refers to humans as well as non-humans, including, for
example, mammals, birds, reptiles, amphibians, and fish. For
instance, the non-humans may be mammals (e.g., a rodent, a mouse, a
rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). In
certain embodiments parenteral routes are desirable since they
avoid contact with the digestive enzymes that are found in the
alimentary canal. According to such embodiments, inventive
compositions may be administered by injection (e.g., intravenous,
subcutaneous or intramuscular, intraperitoneal injection),
rectally, vaginally, topically (as by powders, creams, ointments,
or drops), or by inhalation (as by sprays).
[0088] In a particular embodiment, disclosed nanoparticles may be
administered to a subject in need thereof systemically, e.g., by IV
infusion or injection.
[0089] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In one
embodiment, the inventive conjugate is suspended in a carrier fluid
comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)
TWEEN.TM. 80. The injectable formulations can be sterilized, for
example, by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0090] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the encapsulated or unencapsulated conjugate is mixed with at least
one inert, pharmaceutically acceptable excipient or carrier such as
sodium citrate or dicalcium phosphate and/or (a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, (e) solution retarding agents such as paraffin, (f)
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay, and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets, and pills, the dosage
form may also comprise buffering agents.
[0091] Disclosed nanoparticles may be formulated in dosage unit
form for ease of administration and uniformity of dosage. The
expression "dosage unit form" as used herein refers to a physically
discrete unit of nanoparticle appropriate for the patient to be
treated. For any nanoparticle, the therapeutically effective dose
can be estimated initially either in cell culture assays or in
animal models, usually mice, rabbits, dogs, or pigs. An animal
model may also be used to achieve a desirable concentration range
and route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
Therapeutic efficacy and toxicity of nanoparticles can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED.sub.50 (the dose is
therapeutically effective in 50% of the population) and LD.sub.50
(the dose is lethal to 50% of the population). The dose ratio of
toxic to therapeutic effects is the therapeutic index, and it can
be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions which exhibit large therapeutic indices may be useful
in some embodiments. The data obtained from cell culture assays and
animal studies can be used in formulating a range of dosage for
human use.
[0092] In an exemplary embodiment, a pharmaceutical composition is
disclosed that includes a plurality of nanoparticles each
comprising a therapeutic agent and a pharmaceutically acceptable
excipient.
[0093] In some embodiments, a composition suitable for freezing is
contemplated, including nanoparticles disclosed herein and a
solution suitable for freezing, e.g., a sugar (e.g. sucrose)
solution is added to a nanoparticle suspension. The sucrose may,
e.g., act as a cryoprotectant to prevent the particles from
aggregating upon freezing. For example, provided herein is a
nanoparticle formulation comprising a plurality of disclosed
nanoparticles, sucrose and water; wherein, for example, the
nanoparticles/sucrose/water are present at about
5-10%/10-15%/80-90% (w/w/w).
[0094] In an embodiment, provided herein is a pharmaceutical
aqueous suspension comprising a plurality of nanoparticles, for
example, as disclosed herein, having a glass transition temperature
between about 37.degree. C. and about 50.degree. C., or about
37.degree. C. and about 39.degree. C. in said suspension.
Methods of Treatment
[0095] In some embodiments, therapeutic particles disclosed herein
may be used to treat, alleviate, ameliorate, relieve, delay onset
of, inhibit progression of, reduce severity of, and/or reduce
incidence of one or more symptoms or features of a disease,
disorder, and/or condition. For example, disclosed therapeutic
particles, that include epothilone, e.g., epothilone B, may be used
to treat cancers such as breast, prostate, colon, glioblastoma,
acute lymphoblastic leukemia, osteosarcoma, non-Hodgkin's lymphoma,
or lung cancer such as non-small cell lung cancer in a patient in
need thereof.
[0096] Disclosed methods for the treatment of cancer (e.g. breast
or prostate cancer) may comprise administering a therapeutically
effective amount of the disclosed therapeutic particles to a
subject in need thereof, in such amounts and for such time as is
necessary to achieve the desired result. In certain embodiments of
the present invention a "therapeutically effective amount" is that
amount effective for treating, alleviating, ameliorating,
relieving, delaying onset of, inhibiting progression of, reducing
severity of, and/or reducing incidence of one or more symptoms or
features of e.g. a cancer being treated.
[0097] Also provided herein are therapeutic protocols that include
administering a therapeutically effective amount of an disclosed
therapeutic particle to a healthy individual (i.e., a subject who
does not display any symptoms of cancer and/or who has not been
diagnosed with cancer). For example, healthy individuals may be
"immunized" with an inventive targeted particle prior to
development of cancer and/or onset of symptoms of cancer; at risk
individuals (e.g., patients who have a family history of cancer;
patients carrying one or more genetic mutations associated with
development of cancer; patients having a genetic polymorphism
associated with development of cancer; patients infected by a virus
associated with development of cancer; patients with habits and/or
lifestyles associated with development of cancer; etc.) can be
treated substantially contemporaneously with (e.g., within 48
hours, within 24 hours, or within 12 hours of) the onset of
symptoms of cancer. Of course individuals known to have cancer may
receive inventive treatment at any time.
[0098] In other embodiments, disclosed nanoparticles may be used to
inhibit the growth of cancer cells, e.g., breast cancer cells. As
used herein, the term "inhibits growth of cancer cells" or
"inhibiting growth of cancer cells" refers to any slowing of the
rate of cancer cell proliferation and/or migration, arrest of
cancer cell proliferation and/or migration, or killing of cancer
cells, such that the rate of cancer cell growth is reduced in
comparison with the observed or predicted rate of growth of an
untreated control cancer cell. The term "inhibits growth" can also
refer to a reduction in size or disappearance of a cancer cell or
tumor, as well as to a reduction in its metastatic potential.
Preferably, such an inhibition at the cellular level may reduce the
size, deter the growth, reduce the aggressiveness, or prevent or
inhibit metastasis of a cancer in a patient. Those skilled in the
art can readily determine, by any of a variety of suitable indicia,
whether cancer cell growth is inhibited.
[0099] Inhibition of cancer cell growth may be evidenced, for
example, by arrest of cancer cells in a particular phase of the
cell cycle, e.g., arrest at the G2/M phase of the cell cycle
Inhibition of cancer cell growth can also be evidenced by direct or
indirect measurement of cancer cell or tumor size. In human cancer
patients, such measurements generally are made using well known
imaging methods such as magnetic resonance imaging, computerized
axial tomography and X-rays. Cancer cell growth can also be
determined indirectly, such as by determining the levels of
circulating carcinoembryonic antigen, prostate specific antigen or
other cancer-specific antigens that are correlated with cancer cell
growth Inhibition of cancer growth is also generally correlated
with prolonged survival and/or increased health and well-being of
the subject.
[0100] Other methods contemplated herein include methods of
treating neurodegenerative ailments such as Alzheimer's disease in
a patient in need thereof that include administering a disclosed
nanoparticle, e.g. a disclosed nanoparticle having epothilone
D.
EXAMPLES
[0101] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention in any way.
Example 1
Preparation of PLA-PEG
[0102] The synthesis is accomplished by ring opening polymerization
of d,l-lactide with .alpha.-hydroxy-.omega.-methoxypoly(ethylene
glycol) as the macro-initiator, and performed at an elevated
temperature using Tin (II) 2-Ethyl hexanoate as a catalyst, as
shown below (PEG Mn.apprxeq.5,000 Da; PLA Mn.apprxeq.16,000 Da;
PEG-PLA M.sub.n.apprxeq.21,000 Da).
##STR00003##
[0103] The polymer is purified by dissolving the polymer in
dichloromethane, and precipitating it in a mixture of hexane and
diethyl ether. The polymer recovered from this step is dried in an
oven.
Example 2
Nanoparticle Preparation
[0104] Epothilone B nanoparticles were produced using the following
formulations: [0105] 10% (w/w) theoretical drug [0106] 90% (w/w)
Polymer-PEG, 16-5 PLA-PEG or 50-5 PLA-PEG [0107] % Total Solids=20%
[0108] Solvents: 21% benzyl alcohol, 79% ethyl acetate (w/w) For a
1 gram batch size, 100 mg of drug was mixed with 900 mg of
Polymer-PEG: 16-5 or 50-5 PLA-PEG.
[0109] Epothilone B nanoparticles were produced as follows. In
order to prepare a drug/polymer solution, 100 mg of epothilone B
was added to a 7 mL glass vial along with 3.16 g of ethyl acetate.
The mixture was vortexed until the drug was mostly dissolved.
Subsequently, 0.840 g of benzyl alcohol was added to the glass vial
and vortexed until the drug was completely dissolved. Lastly, 900
mg of polymer-PEG was added to the mixture and vortexed until
everything was dissolved.
[0110] An aqueous solution for either a 16-5 PLA-PEG formulation or
a 50-5 PLA-PEG formulation was prepared. The 16-5 PLA-PEG
formulation contained 0.1% Sodium Cholate, 2% benzyl alcohol, and
4% ethyl acetate in water. Specifically, 1 g of sodium cholate and
939 g of DI water were added to a 1 L bottle and mixed using a stir
plate until they were dissolved. Subsequently, 20 g of benzyl
alcohol and 40 g of ethyl acetate were added to the sodium
cholate/water mixture and mixed using a stir plate until all were
dissolved. The 50-5 PLA-PEG formulation contained 5% Sodium
Cholate, 2% benzyl alcohol, and 4% ethyl acetate in water.
Specifically, 50 g sodium cholate and 890 g of DI water were added
to a 1 L bottle and mixed using a stir plate until they were
dissolved. Subsequently, 20 g of benzyl alcohol and 40 g of ethyl
acetate were added to the sodium cholate/water mixture and mixed
using a stir plate until all were dissolved.
[0111] An emulsion was formed by combining the organic phase into
the aqueous solution at a ratio of 5:1 (aqueous phase:oil phase).
The organic phase was poured into the aqueous solution and
homogenized using a rotor stator homogenizer for 10 seconds at room
temperature to form a coarse emulsion. The solution was
subsequently fed through a high pressure homogenizer (110S), with
one interaction chamber, 100 .mu.m Z-chamber. For the 16-5 PLA-PEG
formulation, the pressure was set to 9900 psi for two discreet
passes to form the nanoemulsion. For the 50-5 PLA-PEG formulation,
the pressure was set to 9900 psi for two discreet passes and then
increased to 13200 psi for two additional passes.
[0112] The emulsion was quenched into cold DI water at
<5.degree. C. while stirring on a stir plate. The ratio of
Quench to Emulsion was 8:1.35% (w/w) Tween 80 in water was then
added to the quenched emulsion at a ratio of 25:1 (Tween
80:drug).
[0113] The nanoparticles were concentrated through tangential flow
filtration (TFF) followed by diafiltration to remove solvents,
unencapsulated drug and solubilizer. A quenched emulsion was
initially concentrated through TFF using a 300 KDa Pall cassette (2
membrane) to an approximately 100 mL volume. This was followed by
diafiltration using approximately 20 diavolumes (2 L) of cold DI
water. The volume was minimized by adding 100 mL of cold water to
the vessel and pumping through the membrane for rinsing.
Approximately 100-180 mL of material were collected in a glass
vial. The nanoparticles were further concentrated using a smaller
TFF to a final volume of approximately 10-20 mL.
[0114] In order to determine the solids concentration of unfiltered
final slurry, a volume of final slurry was added to a tared 20 mL
scintillation vial and dried under vacuum on lyo/oven. Subsequently
the weight of nanoparticles was determined in the volume of the
dried down slurry. Concentrated sucrose (0.666 g/g) was added to
the final slurry sample to attain a final concentration of 10%
sucrose.
[0115] In order to determine the solids concentration of 0.45 .mu.m
filtered final slurry, a portion of the final slurry sample was
filtered before the addition of sucrose using a 0.45 .mu.m syringe
filter. A volume of the filtered sample was then added to a tared
20 mL scintillation vial and dried under vacuum on lyo/oven. The
remaining sample of unfiltered final slurry were frozen with
sucrose.
Example 3
Particle Size and Drug Load Analysis
[0116] Particle size was analyzed by two techniques--dynamic light
scattering (DLS) and laser diffraction. DLS was performed using a
Brookhaven ZetaPals instrument at 25.degree. C. in dilute aqueous
suspension using a 660 nm laser scattered at 90.degree. and
analyzed using the Cumulants and NNLS methods. Laser diffraction
was performed with a Horiba LS950 instrument in dilute aqueous
suspension using both a HeNe laser at 633 nm and an LED at 405 nm,
scattered at 90.degree. and analyzed using the Mie optical model.
The output from the DLS was associated with the hydrodynamic radius
of the particles, which includes the PEG "corona", while the laser
diffraction instrument is more closely associated with the
geometric size of the PLA particle "core".
[0117] Table 1 gives the particle size and drug load of the
particles described above.
TABLE-US-00001 TABLE 1 Particle EpoB Load Size Formulation
Description (%) (nm) 16/5 PLA/PEG 20% solids, 2 passes at 9900 psi
2.3 91 50/5 PLA/PEG 20% solids, 2 passes at 9900 psi 1.6 174 and 2
passes at 13200 psi
Example 4
In Vitro Release
[0118] To determine the in vitro release of epothilone B from the
nanoparticles, the nanoparticles were suspended in PBS release
media and incubated in a water bath at 37.degree. C. Samples were
collected at specific time points. An ultracentrifugation method
was used to separate released drug from the nanoparticles.
[0119] FIG. 3 shows the results of an in vitro release study on the
16-5 PLA-PEG and 50/5 PLA/PEG formulations. Data shows 100% release
of Epo B from the 16/5 PLA/PEG formulation after one hour. The 50/5
PLA/PEG formulation is a slower releasing formulation with 50%
release at 1 hour, 60% release at 2 hours, 70% release at 4 hours,
and greater than 80% drug release at 24 hours. The two formulations
demonstrate the ability to encapsulate epothilone B into
nanoparticles and the ability to impact in vitro release through
the selection of the polymer type used in the formulation.
Example 5
Emulsion Preparation
[0120] A general emulsion procedure for the preparation of drug
loaded nanoparticles in aqueous suspension (10 wt. % in sucrose,
3-5 wt. % polymeric nanoparticles containing about 10 wt. % drug
with respect to particle weight) is summarized as follows. An
organic phase is formed composed of 30% solids (wt %) including 24%
polymer and 6% active agent. The organic solvents are ethyl acetate
(EA) and benzyl alcohol (BA), where BA comprises 21% (wt %) of the
organic phase. The organic phase is mixed with an aqueous phase at
approximately a 1:2 ratio (oil phase:aqueous phase) where the
aqueous phase is composed of 0.25% sodium cholate, 2% BA, and 4% EA
(wt %) in water. The primary emulsion is formed by the combination
of the two phases under simple mixing or through the use of a rotor
stator homogenizer. The primary emulsion is then formed into a fine
emulsion through the use of a high pressure homogenizer. The fine
emulsion is then quenched by addition to a chilled quench
(0-5.degree. C.) of deionized water under mixing. The
quench:emulsion ratio is approximately 10:1. Then, a solution of
35% (wt %) of Tween-80 is added to the quench to achieve
approximately 4% Tween-80 overall. The nanoparticles are then
isolated and concentrated through
ultrafiltration/diafiltration.
[0121] In an exemplary procedure to make fast-releasing
nanoparticles with suppressed T.sub.g, 50% of the polymer is
polylactide-poly(ethylene glycol) diblock copolymer (PLA-PEG; 16
kDa-5 kDa) while 50% of the polymer is poly(D,L-lactide) (PLA; 8.51
kDa).
[0122] In an exemplary procedure to make normal-releasing
nanoparticles with augmented T.sub.g, 100% of the polymer is
polylactide-poly(ethylene glycol) diblock copolymer (PLA-PEG; 16
kDa-5 kDa).
[0123] In an exemplary procedure to make slow-releasing
nanoparticles with augmented T.sub.g, 50% of the polymer is
polylactide-poly(ethylene glycol) diblock copolymer (PLA-PEG; 16
kDa-5 kDa) while 50% of the polymer is poly(D,L-lactide) (PLA; 75
kDa).
Example 6
Animal Studies
[0124] FIG. 4 depicts the pharmacokinetics of slow release and fast
release nanoparticles as in Example 5, having epothilone B.
Sprague-Dawley rats (n=3/group) were administered a dose of 0.5
mg/kg.
EQUIVALENTS
[0125] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
INCORPORATION BY REFERENCE
[0126] The entire contents of all patents, published patent
applications, websites, and other references cited herein are
hereby expressly incorporated herein in their entireties by
reference.
* * * * *